In vitro fertilization (IVF) of human oocytes is a widely practiced medical technique used to overcome various forms of female and male infertility. As a result of this procedure, tens of thousands of babies have been born world-wide to previously infertile couples. Although first practised to treat women with blocked Fallopian tubes, IVF has been successfully applied to treat other types of infertility. However, one application that has not been found to be possible is IVF using cryopreserved immature oocytes.
Cryopreservation of sperm is a well-established technique which allows the preservation of fertility for men who have medical conditions which will result in the loss of their ability to produce healthy, functional sperm. The availability of cryopreserved sperm also greatly facilitates the availability of sperm from anonymous donors to be used for therapeutic donor insemination. However, such possibilities are not available for women who are about to lose their fertility, or who require donor oocytes because very few unfertilized oocytes have been successfully cryopreserved and subsequently fertilized (reviewed by Bernard and Fuller, Human Reprod. Update 2: 193-207, 1996; Chen, Lancet i: 884-886, Apr. 19, 1986; van Uem et al.,Lancet i: 752-753, Mar. 28, 1987). This problem is unique to unfertilized oocytes, since fertilized oocytes and embryos are routinely successfully cryopreserved for future use.
The ovaries of most women in the childbearing years contain thousands of immature oocytes. However, immature oocytes cannot be fertilized until they have undergone maturation. In each menstrual cycle, usually only one oocyte matures to be released from the ovary at the time of ovulation, and possibly fertilized. When IVF was first performed, the one mature unfertilized oocyte was removed from the ovary just prior to ovulation. It was fertilized in a laboratory dish (in vitro) and the resulting embryo was transferred back to the woman's uterus. However, it was found that if more oocytes were available for fertilization, there were more embryos available for transfer to the uterus and this significantly increased the pregnancy rate. Therefore, the current clinical practice involves giving patients hormone injections in order to induce the maturation of approximately twenty oocytes. Just prior to ovulation, the mature oocytes are removed from the ovaries using an ultrasonographically guided probe inserted in the vagina. The oocytes are then placed in appropriate culture medium and mixed with either partner or donor sperm. After twenty-four hours, the oocytes are evaluated for signs of fertilization and fertilized oocytes are kept in culture for 2 to 5 more days, to allow them to develop into embryos. Generally, up to 3 embryos are transferred to the woman's uterus. Any additional embryos may be cryopreserved for future use (Wood, C. and Trounson, A. eds "Clinical in Vitro Fertilization" Berlin: Springer-Verlag 1989).
The administration of hormone injections to induce the maturation of many oocytes simultaneously is known as controlled ovarian hyperstimulation (COH). The advantage of COH is the availability of many more mature oocytes for fertilization, which increases the chances of pregnancy. However, it is necessary that the effects of the injections are closely monitored by daily ultrasound examinations of the ovaries and blood hormone measurements, since excessive ovarian stimulation may cause ovarian hyperstimulation syndrome (OHSS), which is a serious and potentially fatal condition.
Some disadvantages of COH are listed as follows:
1. The risk of OHSS. PA1 2. The requirement for twice daily hormone injections and daily ultrasound examinations and blood tests. Each of these requirements is inconvenient, uncomfortable and expensive. PA1 3. The timing of oocyte retrievals and fertilization is determined by the patient's response to COH. Accordingly, patients and health care providers must be available seven days a week. PA1 4. Some patients fail to produce an adequate number of oocytes, despite the administration of large amounts of hormones. PA1 1. At a time between day 3 and day 5 of the menstrual cycle, ovarian follicles from 2 to 10 mm in diameter are identified using transvaginal ultrasonography and by use of a transvaginal probe, oocytes are aspirated from these follicles into a physiologically acceptable culture medium containing an effective concentration of dbcAMP to inhibit maturation. PA1 2. The immature oocytes are cultured for 5 to 7 days which allows them to grow and develop to be subsequently developmentally competent, without undergoing maturation. PA1 3. The oocytes are then rinsed in, and transferred to, a physiologically acceptable culture medium free of dbcAMP and allowed to mature. PA1 4. Forty-eight hours later, the oocytes are examined and those which are mature are transferred to culture dishes containing the selected and desired sperm. After 4 hours, oocytes are transferred to fresh culture medium. PA1 5. The next day, oocytes are examined for signs of fertilization. Fertilized oocytes are monitored as they undergo embryonic development. PA1 6. Generally, one to three embryos are transferred to a uterus between 2 and 5 days after fertilization. PA1 7. Any additional embryos may be cryopreserved for future use, as is standard practice.
An alternative way of obtaining mature oocytes for IVF is to remove immature oocytes from the ovaries and to allow them to mature in vitro. Mammalian oocytes, including human oocytes, are known to undergo maturation in vitro. In the case of mice, cattle and other mammals, in vitro matured oocytes have been fertilized in vitro and given rise to normal healthy offspring when embryos were transferred to an appropriate uterus (Schroeder and Eppig 1984 Dev. Biol. 102:493; Sirar et al. 1988, Biol.Reprod. 39:546). A few similar attempts have been carried out in humans, resulting in pregnancies in approximately 2% of patients treated (Cha et al., 1991, Fertil. Steril. 55:109; Trounson et al., 1994, Fertil. Steril. 62:353; Barnes et al., Hum. Reprod. 1995, 10:101; Russell et al., 1996, Human Reprod. 11: Abstract Book 1, p.2).
Mammalian oocytes, including human oocytes, undergo spontaneous maturation in vitro when removed from the ovaries and cultured under physiological conditions, with human immature oocytes becoming mature within 48 hours. Previous investigators have removed oocytes from the ovaries 3-8 days prior to the expected time of ovulation, allowed the oocytes to mature in vitro for 48 hours, fertilized them, and approximately 48 hours after fertilization, transferred the resulting embryos to the uterus. However, the timing of fertilization and embryo transfer are generally not optimal for the following reasons.
1. Oocyte quality
Ovaries contain thousands of immature oocytes. In any given month, early in the menstrual cycle, several oocytes begin to grown in preparation for undergoing maturation and becoming developmentally competent, i.e. competent to be fertilized and develop into a healthy mammal. By approximately the fifth to seventh day of the cycle, one oocyte becomes dominant and continues to grow while the others are induced to degenerate. Once an oocyte becomes dominant, it grows and undergoes metabolic changes for approximately one week prior to becoming mature at the time of ovulation. Oocytes that do not undergo this growth phase will mature in vitro and can be fertilized, but are less likely to be developmentally competent. Therefore, the optimal time to obtain the largest number of immature oocytes is early in the cycle before any oocytes have begun to degenerate. However, unfortunately, oocytes removed early in the menstrual cycle and matured in vitro, are less likely to be developmentally competent.
2. Timing of Embryo Transfer
When immature oocytes are obtained early in the menstrual cycle and allowed to mature, they are fertilized earlier in the cycle than would normally occur. This results in the formation of embryos which are then placed in the uterus at a time in the menstrual cycle which is earlier than that which would occur as a result of fertilization in a natural cycle. The endometrium, the lining of the uterus, undergoes growth and development during the menstrual cycle, in preparation for embryo implantation. If the embryo arrives in the uterus too early, the endometrium is not adequately developed, and implantation cannot occur. This problem has been addressed by preparing the endometrium to be ready sooner by the administration of hormones. However, this approach may not provide the optimal conditions for embryo implantation.
One solution to these problems would be to delay fertilization of mature oocytes until a more physiological time in the menstrual cycle. However, mature eggs that are kept in culture for more than 24 hours begin to deteriorate and after 48 hours can no longer be fertilized.
Another solution is to maintain oocytes in culture in the immature state. In the ovary, oocytes remain in the immature state until stimulated to mature by the hormone, luteinizing hormone (LH). Therefore, it seems that there is an inhibitor in the ovary which prevents the premature maturation of oocytes which occurs spontaneously in vitro. This inhibitor has been named oocyte maturation inhibitor (OMI) but its molecular nature has not yet been identified, despite more than 20 years of research. However, it is known that compounds which increase the levels of oocyte cyclic adenosine monophosphate (cAMP), a ubiquitous intracellular signalling molecule, prevent oocyte maturation in vitro (Cho et al., 1974, J. Exp. Zool. 187:383). Accordingly, including a compound which increases levels of oocyte cAMP in the culture medium, allowed immature oocytes from immature mice to grow in vitro to a size at which they were developmentally competent (Chesnel et al., 1994, Dev. Biol., 161:285).
The technical ability to successfully and controllaby mature oocytes in vitro has a direct application to the problem of mature oocyte cryopreservation. To-date, attempts at oocyte cryopreservation have involved the use of mature oocytes, since these are the oocytes that can be successfully fertilized in vitro. However, mature oocytes do not survive cryopreservation well because mature oocytes are at the metaphase stage of the cell cycle wherein they contain a microtubule spindle along which the paired chromosomes are arranged. At the time of fertilization, the chromosomes separate, one member of each pair travelling in opposite directions along tracks provided by the spindle fibres. Any disruption of the spindle can lead to incorrect chromosome separation, which is severely detrimental or lethal to a cell. Freezing is known to cause the disappearance of the spindle and disruption of the chromosome arrangement. The spindle reforms upon thawing, but the chromosomes may no longer be correctly arranged. Disruption of the spindle impairs cleavage divisions, which are essential to normal embryonic development. The disruption of the spindle caused by cryopreservation may well explain the poor results seen with cryopreserved oocytes. In contrast, immature oocytes, at the prophase stage of the cell cycle, do not contain a microtubule spindle. Therefore, the cryopreservation of immature oocytes eliminates the problem of damage to the spindle. However, if immature oocytes are to be cryopreserved, it is necessary to have a technique whereby after thawing they can be successfully and controllably matured and fertilized in vitro.
However, to-date, there remains a need for a clinical IVF procedure which is more convenient, less potentially harmful and less expensive for mammals, particularly humans. The present invention provides such a technique.